In the late 1980s, it was discovered that direct intramuscular (i.m.) injection of lipid-DNA complexes results in measurable protein expression, and that “naked” plasmid DNA (pDNA) is taken up and expressed in muscle to a greater extent than lipid-DNA complexes (Felgner, Scientific American, 276(6), 102-106 (1997)). Later, it was shown that cationic lipid-protein mixtures afforded greater immune responses after i.m. injection than protein alone (Brunel, Vaccine 17, 2192-2203 (1999).
One of the first applications of pDNA injection technology was the induction of an immune response. In 1991, it was first reported that mice could be immunized against HIV gp120 by i.m. vaccination with gp120 plasmid DNA (Felgner et al., Nature, 349, 351-352 (1991)), and that mice could be protected from a lethal challenge of influenza virus after DNA immunization with influenza nucleoprotein (NP) antigen. Protection obtained after immunization with the highly conserved NP antigen extended across 2 different viral strains (Ulmer et al., Current Opinions In Immunology, 8, 531-536 (1996)). Numerous publications in the field of polynucleotide-based vaccination followed thereafter (e.g., Boyer et al., J. Med. Primatology, 25(3), 242-250 (1996); Boyer et al., Nature Medicine, 3(5), 526-532 (1997); Davis et al., Vaccine, 15(8), 849-852 (1997); Wang et al., Vaccine, 15(8), 821-825 (1997); Agadjanyan et al., Current Topics In Microbiology And Immunology, 226, 175-192 (1998); Heppell et al., Fish & Shellfish Immunology, 8(4), 271-286 (1998); Lodmell et al., Nature Medicine, 4(8), 949-952 (1998); Vanderzanden et al., Virology, 246(1), 134-144 (1998)).
A major problem frequently encountered in the course of polynucleotide-based vaccination is insufficient or suboptimal humoral response. Often, the antigens or immunogens encoded by the polynucleotide are expressed in vivo, but they are not sufficiently immunogenic to raise the antibody titer in the organism to sufficient levels to provide protection against subsequent challenge and/or to maintain the potential for generating therapeutically active antibody levels over extended time periods. To obtain a stronger humoral and/or cellular response, it is common to administer such vaccines in an immunogenic composition containing an adjuvant, a material which enhances the immune response of the patient to the vaccine. Adjuvants are useful generally for improving the immune response of an organism to a particular immunogen and are commonly included in vaccine compositions to increase the amount of antibodies produced and/or to reduce the quantity of immunogen and the frequency of administration.
A variety of adjuvants have been reported to effect differing levels of immune response enhancement to polynucleotide-based vaccination. Examples of such adjuvant materials include semi-synthetic bacterial cell wall-derived mono-phosphoryl lipid A (Sasaki, S., et al., Infection and Immunity 65(9), 3250-3258 (1997)), small molecule immunostimulators (Sasaki, S., et al., Clin. Exp. Immunol. 111, 30-35 (1998)), and saponins (Sasaki, S., et al., J. Virology 72(6), 4391-4939 (1998)). The immune response from i.m. pDNA vaccination has also been enhanced through the use of cationic lipids (Ishii, N., et al., Aids Res. Hum. Retroviruses 13(16), 1421-1428 (1997)), Okada, E., et al., J Immunology 159, 3638-3647 (1997); Yokoyama, M., et al., FEMS Immunol. Med. Microbiol. 14, 221-230 (1996); Gregoriadis, G., et al., FEBS Letters 402, 107-110 (1997); Gramzinski, R. A., et al., Molecular Medicine 4, 109-118 (1998); Klavinskis, L. S., et al., Vaccine 15(8), 818-820 (1997); Klavinskis, L. S., et al., J. Immunology 162, 254-262 (1999); Etchart, N., et al., J. Gen. Virology 78, 1577-1580 (1997); Norman, J., et al., in Methods in Molecular Medicine, Vol. 29; DNA Vaccines: Methods and Protocols, D. B. Lowrie and R. Whalen, eds., Chapter 17, pp. 185-196 (1999)). Cationic lipids were originally studied to enhance delivery of pDNA into cells in vitro; however, further development has led to successful specific applications of protein delivery in vivo (Wheeler, C. J., et al., Proc. Nail. Acad. Sci. USA 93, 11454-11459 (1996); Stephan, D. J., et al., Human Gene Therapy 7, 1803-1812 (1996); DeBruyne, L. A., et al., Gene Therapy 5, 1079-1087 (1998)). Accordingly, such cationic lipids may be useful for vaccine applications by enhancing delivery of the pDNA into the cells responsible for giving rise to the humoral arm of the immune response, thereby increasing antibody titer levels.
Commonly used adjuvants, such as Alum, show low levels of immune response enhancement for vaccination (typically less than 3-fold) and possess undesirable toxicological and manufacturing profiles. In addition, cationic lipids used previously for vaccination show only low levels of humoral enhancement. Thus, there is a need for more adjuvant compositions useful for enhancing the immune response of vertebrates to immunization, especially to pDNA vaccination.